Since the early 1980's scientists have been seeking new and better methods for transforming plants of all type. Initially, dicots were more readily transformed and monocots were fairly difficult. However, in the last ten years methods of transforming both types of plants have been achieved. In light of these achievements the choice of method for the transformation of plant cells tends to be limited to those which are convenient for the target plant type. As a generalisation, research in transformation has focused on new techniques which improve the usefulness of the transformation methods. Several transformation methods which have been available for introduction of foreign DNA include: Agrobacterium technology (U.S. Pat. No. to 5,591,616 to Japan Tobacco); electroporation technology (U.S. Pat. No. 5,679,588 to PGS), and microinjection. This last method known as “microinjection” is where a DNA construct is injected from a hollow needle into a target cell. A variant of that procedure is the rupturing of the cell wall with a needle, the DNA being added to the surrounding medium and allowed to diffuse into the cell through the break in the cell wall. This is known as “micropricking”. Both of these procedures require a high degree of manipulative skill by the operator and are very time consuming.
The most well known transformation approach may be the biolistic bombardment. In this method microparticles coated with DNA are accelerated and blasted into cells (U.S. Pat. No. 4,945,050 to Cornell and U.S. Pat. No. 5,538,877 to Dekalb). This approach abandons the high precision of targeting which is inherent in microinjection and micropricking, in favour of a rapid “shotgun” approach which blasts large numbers of cells in a short time, giving a larger number of putative transformants for screening.
In such an approach, tungsten or gold microspheres, coated with DNA are fired at target tissue at very high velocity, for example under propulsion of an explosive charge. One problem with this technique is the effect of the blast on the target tissue.
The most closely related technology to the present invention is another transformation method that involves the use “whiskers” to transform (U.S. Pat. Nos. 5,302,523 and 5,463,765 to Zeneca and PCT/US99/01815 to Dow Agrosciences LLC). In this approach there was provided a method of transforming cells comprising contacting the cells with a multiplicity of needle-like bodies so that the cells are impaled upon the bodies, transforming DNA being either surface-bound to the projections or present in a liquid medium in contact therewith.
In one embodiment a quantity of the needle-like bodies was added to a liquid suspension of the cells to be transformed and the mixture agitated, for example by stirring, so that the moving cells and bodies interact resulting in penetration of the cell wall of the cells.
However, this whisker method lacked efficiency. Even when the first applications were filed it was realized that efficiency could come from all different aspects of whiskering.
At that time the Zeneca application stated was stated that, It has been found that the efficiency of DNA delivery varies according to the conditions; it is affected by several factors including the following: vortex time; cell suspension type (variation also found by H. F. Kaeppler et al, 1990, Plant Cell Reports, 9, 415-418); cell suspension age; osmolarity of culture medium; type of fibres; number of fibres present; type of DNA construct; concentration of DNA. Other factors which may affect DNA delivery include: the physical mixing methods used; the size, shape and uniformity of the fibres; the topology of the DNA (eg. linear, supercoiled); the presence of “carrier” DNA alongside the transforming DNA.
What was not discovered or suggested was that a change in the agatition step would substantially reduce, by a third at least, the time and effort needed to complete whisker mediated transformation of cells.
Another method used through out PCT/US 99/01815 to Dow Agrosciences LLC comprised mixing the DNA and fibre suspension, then adding this mixture to the target tissue. The final mixture was vortexed together. The cells were then incubated, and tested for expression of recombinant DNA and regenerated into transgenic plants.
Throughout the application underlying PCT/US 99/01815 the examples are attempting to optimize the production of small quantities of transformants using the Whisker transformation process through, for example, use of osmotic treatments, testing vessels and varying agitation (vortexing) time (see Table 9 in PCT/US 99/01815). In table nine the effect of vortexing time on the transformation of rice showed that 15 seconds of vortexing resulted in twice as much Gus expression as did 120 seconds of vortexing. The expression of the transformed gene in the small microfuge tubes decreased with each increase in vortexing time. Thus increased agitation by vortexing led to less positive transformation results. Therefore altering the type of agitation from vortexing to a more robust, or violent agitation method seemed an unlikely way of increasing production of transformants.
Thus there remained a need for a new technique within a whisker method that permits large amounts of target tissue to be transformed more effectively.
Additionally, there remains a need for an improvement in the whisker method that allows previously incompetent tissue (or tissue with very low transformant yield) to be more effectively transformed with whiskers.
Furthermore, there remained a need for a method that more effectively accesses whiskers and DNA to most of the surface area of the target tissue thus whereby tissues are more effectively transformed.
Additionally, there was a need for an improved whisker method that could be used with all of the target tissues that the regular method could be employed with such as type I, type II, type III callus or explants or scutella or zygotic embryos, protoplasts, hypcotyl or cotyledon derived callus, stomato cells, germline tissue, cell suspensions cultures and elite germplasm.
Although the whisker technology has undergone numerous technical achievements, including use of different types of target tissues like Type I and type two II callus or either elite or non-elite germplasm, forming commercially viable transgenic plants with the whisker method is still a laborious task. The method does not produce as many transformants per experiment as would be desirable when compared to using the gun technique. Whiskers low efficiency rate results in difficulties and added costs.
There remains a need for a modified method that will yield larger quantities of putative transformants than presently exists.
There also remains a need for an efficient whiskering method for transforming elite Zea mays germplasm.
There also remains a need for a more time efficient whisker method for transforming target tissue from wheat, barley, rye, soybeans, Brassica species, and grasses including perennial ryegrass, bentgrass and the like.
Additionally, there remains a need for a whisker method that may be employed to transform stomato cells or “guard cells” which are regenerable without the step of forming a protoplast.